FUNDAMENTAL AND APPLIED TOXICOLOGY 3 3 , 1 0 9 - 1 1 9 (1996)
ARTICLE NO. 0148
Comparative Absorption of Lead from Contaminated Soil and Lead
Salts by Weanling Fischer 344 Rats
G. B.
FREEMAN,*
J. A. DiLL,t J. D.
JOHNSON,*
P. J.
KURTZ,*
F.
PARHAM,$ AND
H. B.
MATTHEWS^
*Battelle, 505 King Avenue, Columbus. Ohio 43201; \Bastelle Northwest Laboratories, Richland, Washington 99352; and
tNIEHS, P.O. Box 12233, Research Triangle Park, North Carolina 27709
Received January 9, 1996; accepted May 23, 1996
been previously described and evaluated (ATSDR, 1991).
Extensive literature reports and reviews are available on the
systemic, immunological, neurological, developmental, reproductive, genotoxic, and carcinogenic effects of acute to
chronic lead exposure by the inhalation, oral, and dermal
routes of administration. However, there is still a lack of
information regarding the absorption of lead encountered in
the environment. This is particularly true of lead that may
be consumed by children in the form of dust and dirt originating from soil. Children are a particular concern because they
are both more sensitive to lead intoxication and more readily
absorb ingested lead (ATSDR, 1991).
In assessing potential exposures of young children to lead
in soil, the U.S. Environmental Protection Agency (EPA)
generally relies on the uptake/biokinetic (UBK) model to
predict blood lead levels that will be associated with lead
concentrations in soil (Cohen et ai, 1991; Harley and Kneip,
1985; U.S. EPA, 1989a,b). The default assumption in a recent version of the model is that 30% of soil lead ingested
will be absorbed. This may be an accurate estimate for soils
in which the source of contamination is typical of those
found in some urban areas. However, studies of blood lead
levels in young children in several mining communities have
revealed much lower blood lead levels than predicted by the
UBK model when run using default assumptions (Schoof et
ai, 1993). Due to these findings, recent animal studies have
been initiated to address health risk assessment issues related
to the bioavailability of mining waste lead (Dieter et ai,
1993, 1994; Freeman et ai, 1992, 1994; Ghantous et ai,
1994; Poppenga et ai, 1994; Weis et ai, 1994).
Comparative Absorption of Lead from Contaminated Soil and
Lead Salts by Weanling Fischer 344 Rats. FREEMAN, G. B., DILL,
J. A., JOHNSON, J. D., KURTZ, P. J., PARHAM, F., AND MATTHEWS,
H. B. (1996). Fundam. Appl. Toxicol. 33, 109-119.
A 44-day dosed feed study was performed to compare the bioavailability of lead from contaminated soil versus two lead salts
and the effect of soil on gastrointestinal absorption of ingested
lead. Male Fischer rats (approximately 4 weeks of age) received
lead, 17, 42, or 127 ppm, in the form of lead acetate, lead sulfide,
lead-contaminated soil, or combinations thereof in the diet for 7,
15, or 44 days. Control soil was added to the diets of some animals
to determine how it might alter lead bioavailability. Blood Aaminolevulinic acid dehydratase (A-ALAD) and blood, bone, kidney, and liver lead were determined in groups of animals at each
time-point. Blood A-ALAD was inhibited in a dose-dependent
manner and to the greatest degree in the lead acetate and lead
acetate/control soil groups, followed by the lead sulfide and leadcontaminated soil groups. Bone and tissue lead levels increased in
a dose-dependent manner and were greatest in animals receiving
lead acetate and significantly less in animals receiving lead sulfide
and lead-contaminated soil. Blood lead levels were generally greatest by 7 days and stabilized at lower levels thereafter. Bone lead
concentration-time patterns did not demonstrate the biphasic
change seen with tissues and continued to increase in most treatment groups through the course of the study. The presence of soil
in the diet clearly attenuated the absorption of lead acetate, but
had little effect on the absorption of lead sulfide. Results of these
studies confirm previous observations that lead absorption is
highly dependent on the form of lead ingested and the matrix in
which it is ingested. More important, these studies demonstrate
that lead in soil may be significantly less available than estimated
by current default assumptions and that the presence of soil may
decrease the availability of lead from lead salts on which the default assumptions are based. Results presented here also demonstrate that the weanling rat may represent an appropriate model
that could be used to obtain relatively rapid and economical estimates of the availability of lead in complex matrices such as soil.
The bioavailability of ingested chemicals is classically
estimated from studies in which single or multiple dose(s)
are administered orally followed by periodic determinations
of concentrations in blood until most of the chemical has
been eliminated. The concentration-time profile is then used
to calculate the area under the concentration versus time
curve after oral administration (AUCpJ which is compared
to the area under the concentration versus time curve after
intravenous administration (AUC,V). This approach yields
reliable bioavailability estimates for chemicals or elements
O 19% Society of Toxicology
Studies addressing the overall toxicology of lead and exposure levels associated with adverse health effects have
9
0272-0590/96 $ 18 00
Copyright 1$ 1996 by the Society of Toxicology.
All rights of reproduction in any form reserved
110
FREEMAN ET AL.
that are rapidly and completely eliminated. However, for
elements such as lead that are slowly and incompletely eliminated, this approach requires measuring blood lead concentrations for several months, and possibly years, to adequately
characterize the plasma concentration-time profile. Thus,
an alternative approach of comparing tissue concentrations
following subchronic exposure has been used. This approach
is based on the fact that distribution of lead in the body is
initially dependent on the rate of delivery by the blood to
the various organs and tissues and with time, approximately
4 weeks, a steady state is reached (U.S. EPA, 1986). Tissue
and blood concentrations achieved with a soluble lead salt
of known bioavailability can be compared with those
achieved with the lead source of interest to provide a relative
estimate of bioavailability. In some studies, animals have
been dosed daily by the intravenous route to provide estimates of absolute bioavailability (Freeman et ai, 1994).
Differences in the bioavailability of lead from environmental sources have been attributed to factors such as particle size, matrix, and lead species (Chaney et ai, 1989; Davis
et ai, 1992; Ruby et ai, 1992; Steele et ai, 1990). The
solubility of ingested lead phases is dependent on lead mineralogy, encapsulation or armoring of lead phases within alteration and precipitation products and inert minerals (e.g., silicates), and rates of dissolution (kinetic constraints) of the
lead-bearing solids as they traverse the gastrointestinal (GI)
tract (Mushak, 1991).
In the present investigation, a 44-day dosed feed study
was performed to compare the availability of lead from a
sample of lead-contaminated soil with a soluble lead salt,
lead acetate, and a sparingly soluble lead salt, lead sulfide,
as standards. Control soil was carefully selected to match
the geophysical properties of contaminated soil except for
the presence of lead. The effect of the addition of a control
soil to the lead acetate and lead sulfide-dosed feed mixtures
was investigated to determine what effect, if any, other components of soil may have on lead bioavailability. The dosedfeed route was used because oral ingestion of soil used containing lead mine waste is a potential source of lead exposure
for children in several western states. The rat was used as
the test system because it has been shown to be a practical
model to investigate the bioavailability of mining waste lead
(Dieter et ai, 1993; Freeman et ai, 1992, 1994). The weanling rat was selected for this study because food consumption
patterns and stomach pH following food consumption are
similar to those of young children (Chaney, 1991). More
importantly, use of the weanling rat permits determination
of lead uptake at a period of rapid growth and active bone
formation during which children are most vulnerable to lead.
In addition to the use of weanling rats (approximately 4
weeks old), a purified diet low in calcium and fiber was used
to maximize lead absorption. Low dietary calcium increases
lead absorption because calcium and lead are absorbed com-
TABLE 1
Elemental Analysis of Lead-Contaminated and Control Soils"
Analyte
Lead-contaminated soil
Control soil
Aluminum
Antimony
Arsenic
Barium
Beryllium
Cadmium
Calcium
Chromium
Cobalt
Copper
Iron
Lead
Magnesium
Manganese
Mercury
Nickel
Potassium
Selenium
Silver
Sodium
Thallium
Vanadium
Zinc
5,960
7.4
197
612
0.2
51 7
25,800
11.5
5.4
626
52,800
8,440
12,700
6,000
0.97
7.3
1,630
1.0
36.2
368
0.69
24
9,950
7,920
7.4
5.7
109
0.2
1.0
2,450
11.2
5
11.9
12,600
54
2,950
283
0.1
7.8
1,710
0.2
1.2
83 3
0.41
128
109
° Concentrations are in ppm.
petitively (Six and Goyer, 1970; Mahaffey et ai, 1973;
ATSDR, 1991). Regular rat chow attenuates the absorption
of lead by the strong lead binding or precipitative action of
the chow diet. The nutritionally complete purified AIN-76A
diet (Bieri et ai, 1977) not only has less effect on absorption,
but it more closely simulates the low fiber diets of most U.S.
citizens than do rat chow diets. The study was 44 days in
length to provide sufficient time for accumulation of lead in
blood and bone, while balancing the need for exposure during a period of rapid growth.
METHODS
Materials. Lead(II) acetate trihydrate (Aldrich Chemical Company,
Inc , Milwaukee, WI) and lead(ll) sulfide (AESAR/Johnson Matthey, Ward
Hill, MA) were used as standards. Lead-contaminated soil and control soil
were provided by the U S. EPA (Region VIII. Denver, CO). Both soils
were of a generally sandy nature and were composites of numerous residential samples collected at the California Gulch NPL Site (U.S. EPA, 1992).
Analysis of these soils revealed lead concentrations of 8440 ± 201 and 54
± 3 ppm, respectively. Detailed elemental and electron microprobe analyses
of these soils are presented in Tables 1 and 2. respectively.
Test system and animal maintenance. Male Fischer 344 rats (31 days
of age at initiation of dosing) were supplied by Taconic Farms (Germantown, NY). The animals were housed in an environmentally controlled room
where the temperature and relative humidity specifications were 19 to 25°C
and 40 to 10%, respectively. Lighting conditions were set to provide 12 hr
of fluorescent light (6:00 AM to 6:00 PM) followed by 12 hr of darkness
(6:00 PM to 6:00 AM). Animals were individually housed in standard poly-
EVALUATION OF LEAD-CONTAMINATED SOILS
TABLE 2
Occurrence of Lead Minerals in the Lead-Contaminated
and Control Soils
Lead-contaminated
soil (%)
Control soil
(%)
78
4
5
13
—
FePb sulfate
FePb oxide
Cerussite
Pb phosphate
16
2
Tr"
—
Tr
Tr
Tr
0.5
05
3
12
10
40
3
13
Total particles (n)
665
Phase
MnPb oxide
Anglesite
Galena
CuZnPb
PbV oxide
PbOH
PbAsSbCu oxide
Pb silicate
Pb barite
Pb organic
Slag
—
° Tr, trace levels.
carbonate cages, the dimensions of which (width X height X length) were
56 X 32 X 20 cm. Cages contained ALPHA-dri certified alpha cellulose
bedding (Shepherd Specialty Papers, Kalamazoo, MI). All animals were
provided deionized water ad libitum (<0.2 ppm Pb) by glass bottle reservoirs fitted with stainless-steel sipper tubes and were fed ad libitum in glass
feed jars. Untreated control group animals were fed a purified diet [AIN76A complete meal (Zeigler Brothers, Gardners, PA)]. Dosed-feed lead
treatment group animals were fed AIN-76A meal feed into which the appropriate amounts of test article, i.e., test soils, lead acetate, lead sulfide,
or combinations thereof, were incorporated. The AIN-76A complete meal
contained <0.10 /ig Pb/g.
Dosing regimen and administration.
Groups of 30 male rats (approximately 4 weeks of age) received one of the following diets: (A) undosed
AIN-76A meal feed, (B) control soil (<1 ppm lead), (C) lead acetate 17.6
ppm, (D) lead acetate 42.8 ppm, (E) lead acetate 127 ppm, (F) lead acetate
17.6 ppm + 1.5% control soil, (G) lead acetate 42.8 ppm + 1.5% control
soil, (H) lead acetate 127 ppm + 1.5% control soil, (I) lead sulfide 127
ppm, (J) lead sulfide 127 ppm + 1.5% control soil, (K) 0.2% lead-contaminated soil + 1.3% control soil (17.6 ppm lead), (L) 0.5% lead-contaminated
soil + 1.0% control soil (42.8 ppm lead), (M) 1.5% lead-contaminated soil
(127 ppm lead); for up to 44 days. Ten rats/dose group were killed on each
of three euthanization dates (interim euthanizations on Days 7 and 15, and
terminal euthanization on Day 44).
In-life parameters.
Animals were observed twice daily for any signs
of moribundity or mortality. Individual body weights, clinical observations,
and food and water consumption were recorded twice weekly throughout
the study.
Tissue collection, preparation, and analysis. Blood, bone, liver and
kidney samples were collected from rats sacrificed on Study Days 7, 15,
and 44 for determination of lead concentrations. Blood A-aminolevulinic
acid dehydratase (A-ALAD) activity was also determined at each time
point. Rats were anesthetized with a carbon dioxide/oxygen mixture and
bled via cardiac puncture. Blood was collected into vacutainer tubes containing sodium hepann and gently inverted on an aliquot mixer to prevent
clotting prior to analysis. A portion of the blood sample was frozen at
111
-20°C for lead analysis. Another aliquot was used immediately for the AALAD assay. After blood collection, animals were euthanized with carbon
dioxide and the left femur, left kidney, and liver (left lobe) were removed
and frozen at -20°C for lead analyses. Prior to lead determination, femurs
were boiled for ~1 hr in deionized water and residual tissue was removed
with a nylon bristle brush. After rinsing thoroughly, the femurs were dried
to constant weight for 48 hours at 90°C under vacuum and stored until
analyzed.
Analysis for A-ALAD activity was based upon the European Standard
Method (Berlin and Schaller, 1974). A hemolysate was prepared by diluting
whole blood in deionized water, adding an excess of A-ALA, and incubating
the mixture at 37°C for 60 min. The porphobilinogen formed, which was
proportional to the enzyme activity, was determined by adding a modified
Ehrlich's reagent and measuring the color development photometrically.
Results were reported as units per liter, which is equivalent to micromoles of
A-ALA convened to porphobilinogen per minute per liter of erythrocytes.
Samples of blood, bone, or tissue (~1 g) were weighed directly into
acid-leached Teflon liners for Parr bombs (Parr Bomb, Model 4749, PanInstrument Co., Moline, IL). One to 3 ml of Ultrex concentrated HNO 3
(Ultrex, - 7 0 % HNO,, J. T. Baker, Phillipsburg, PA) was added, and the
bombs were sealed and heated at 130 to 150°C for 3 to 4 hr. After cooling,
the bombs were opened, an aliquot of a bismuth solution was pipetted into
the liners as the internal standard, and the contents of the liners were
quantitatively transferred to polypropylene vials and diluted to an appropriate volume with deionized water. Samples were analyzed with a VG
PlasmaQuad Inductively Coupled Plasma-Mass Spectrometer (ICP-MS,
VG Elemental, Winsford, Cheshire, UK). Data were acquired in the scan
mode over the mass range from 203 to 211 atomic mass units (amu) using
three, 30-sec integrations for each sample. The pulse count detector mode
was employed with 25 amu/channel and a dwell time of 320 //sec. Lead
was quantitated at a mass-to-charge ratio {mJz) of 208, corresponding to
the most abundant lead isotope 2O8Pb (abundance 52.3%). The internal standard was measured at mlz 209, corresponding to ^'Bi (abundance 100%).
Spectrometnc standards for lead and bismuth were obtained from the National Institute of Standards and Technology (NIST, Gaithersburg, MD).
The ICP-MS was calibrated daily against freshly made standards prepared
by dilution of NIST spectrometric standards in dilute nitric acid.
The limit of detection (LOD) was defined as the mean system blank
(solvent blank for bone) concentration plus three times the standard deviation of the system or solvent blank. The limit of quantitation (LOQ) was
defined as the mean system or solvent blank concentration plus 10 times
the standard deviation of the system or solvent blank. The LODs were 0.01,
0.3, 0.001, and 0.003 ^g Pb/g and the LOQs were 0.04, 1.1, 0.006, and
0.005 ^jg Pb/g for the blood, bone, liver, and kidney, respectively.
Statistical methods. Statistical analyses of A-ALAD activity and lead
concentrations in blood and bone were performed using a one-way parametric analysis of variance on four subsets of the animal groups: (a) control
and lead acetate groups; (b) control and lead acetate/control soil groups;
(c) control and lead-contaminated soil/control soil groups; (d) control and
all high dose groups. Tabular presentation of A-ALAD and blood and bone
lead data are based on these four subsets.
Each subset of study groups included the two control groups (i.e., the
blank feed and the control soil groups). If the /-"test indicated that significant
differences were present at the 0.05 level between the animal groups, then
Dunnett's test was performed to identify any dose groups that differed
significantly from a specific control group. Because two of the animal
groups were considered control groups, Dunnett's test was performed twice:
once when comparing to the blank feed group and once for the control soil
group. Each application of Dunnett's test was performed at an overall 0.05
significance level.
If significant differences were observed at the 0.05 level by the F test,
then in addition to considering only comparisons to the control groups via
Dunnett's test, it was of interest to consider all pairs of animal groups and
to identify those group pairs which differed significantly Tukey's stu-
112
FREEMAN ET AL.
dentized range test (at an overall 0.05 significance level) was applied to
meet this objective. As a result of testing for normality, lead concentration
data in blood and bone were analyzed after taking natural logarithms. Due
to the type of skewness generally observed in the distribution of concentration data, a normal distribution is usually more appropriate after taking
natural logarithms of the data. No transformation was suggested for AALAD activity data.
RESULTS
In-Life Parameters
All group mean body weight values for lead-exposed animals remained within ±10% of the control (blank feed)
group mean body weight values. Mean body weight gains
indicated that animals were thriving and growing during the
in-life phase while being exposed to lead by dosed-feed
administration. Group mean food consumption values ranged
from 11.4 to 13.3 g/animal/day. Food consumption for all
treated groups was also similar to the control (blank feed)
group suggesting that no palatability problems occurred with
the dosed-feed formulations. Daily exposure index data indicated that approximately proportional increases in the level
of exposure (absolute amount and per body weight) occurred
at the different dose levels for each treatment group. Group
mean water consumption values ranged from 17.1 to 20.0
g/animal/day. Water consumption for all groups was similar
to the control (blank feed) group. Thus, exposure to lead
was proportional to dose and, based on body weight growth
patterns, did not result in overt toxicity.
A-Aminolevulinic
Acid Dehydratase Activity
Significant differences in A-ALAD activity were observed between control (blank feed) and all treated groups
(Table 3). A-ALAD was significantly reduced in the control
soil group relative to blank feed indicating that something
in the soil, possibly something other than lead, inhibits AALAD. Both control groups demonstrated decreases in AALAD with time from Days 7 to 44. A-ALAD activity was
significantly decreased below control levels in all animals
receiving lead in the diet and, in most cases, A-ALAD inhibition increased as the dose increased. The greatest inhibition
was seen with lead acetate, followed by lead sulfide and
lead-contaminated soil. Inhibition in lead-treated animals
versus the control and control soil groups was evident at all
time periods. However, due to the lower background levels
at the later time periods, differences in A-ALAD inhibition
with the various sources of lead were generally greatest at
Day 7 and were largely insignificant by Day 44. Further,
even though the addition of control soil alone to the diet
decreased A-ALAD activity, inclusion of control soil with
the lead salts was not additive. Control soil tended to attenuate inhibition of A-ALAD by lead acetate, but not lead
sulfide.
There is limited information regarding the "normal" lev-
els of A-ALAD in rats in this age range, but the decrease
in control animals between 15 and 44 days is believed to be
related to the physical growth and maturation of rats between
4 and 10 weeks of age. The levels of A-ALAD of young
adult rats in this study (Day 44 values) are in good agreement
with those of F344 rats assayed in a previous study of the
bioavailability of lead from ore (Freeman et ai, 1993). Further, the blood lead concentrations for the control blank
feed group did not attain the putative threshold or minimum
effective concentration (MEC) levels sufficient to inhibit AALAD activity in humans, 0.10 /xg Pb/ml (Klassen et al,
1986). In rats, A-ALAD activity has been reported to remain
unchanged at blood lead concentrations of 0.09 \i% Pb/ml
(Kimber et al, 1986) and 0.11 ^g Pb/ml (Azar et al, 1973).
Thus, the A-ALAD values of the control blank feed animals
are believed to reflect the normal A-ALAD activity for
young immature and young adult male rats.
Blood Lead Levels
Lead was detectable in blood of all animals studied (Table
4). In animals receiving the control diet (blank feed), blood
lead concentrations were highest at the earliest time point,
0.050 //g Pb/g, and decreased with time to 0.012 //g Pb/g at
44 days. Levels of lead observed in animals receiving control
soil in the diet were approximately two to four times higher
than in blank feed controls, but also decreased with time.
Significant differences between the blank feed control group
and treated groups were evident for all groups at all time
points. Blood lead levels observed at later time points were
usually not significantly higher, and in some cases lower, than
those seen at Day 7. This is interpreted as an indication that
steady state may have been achieved by Day 44.
Dose-dependent increases in blood lead were observed in
most animals that received lead acetate. Dose-related trends
were not as evident for animals receiving lead acetate plus
control soil as they were for animals receiving lead acetate
alone, but a general trend was evident. At 15 and 44 days,
the addition of control soil to the diet with lead acetate
resulted in reduced blood lead levels. In many cases, the
decrease was significant. This was not the case with lead
sulfide, where coadministration of control soil appeared to
have little or no significant effect. In contrast to results seen
with lead acetate, increases in blood lead levels with increasing contaminated soil doses were not apparent and showed
no significant decrease with time. Overall, blood lead concentrations were highest following administration of lead
acetate, followed by lead sulfide and lead-contaminated soil.
Bone Lead Concentrations
Concentrations of lead in bone at various times following
each treatment regime are presented in Table 5. Bone lead
concentrations of the blank feed control group ranged from
3.69 to 2.23 ng Pb/g and decreased over the course of the
113
EVALUATION OF LEAD-CONTAMINATED SOILS
TABLE 3
Group Mean A-Aminolevulinic Acid Dehydratase Activity
Mean (SE)'
Dose group
Control (blank feed)
Control soil (1.5%)
Lead acetate (LO)
Lead acetate (ME)
Lead acetate (HI)
Lead acetate + control soil
(1.5%) (LO)
Lead acetate + control soil
(1.5%) (ME)
Lead acetate + control soil
(1.5%) (HI)
Lead sulfide (HI)
Lead sulfide + control soil
(1 5%) (HI)
Lead-contaminated soil (0.2%)
+ control soil (1.3%) (LO)
Lead-contaminated soil (0 5%)
+ control soil (1.0%) (ME)
Lead-contaminated soil (1 5%)
(HI)
Pairs of noncontrol dose groups
significantly different at 0.05
level (Tukey's test)''
Day 7
ppm
<1
17.6
42.8
127
15.5
9.94
5.34"
4.86"
3.57"
Dav 15
11.2
5.72*
4.51
3.68
3.03
(0.6)*
(1.17)
(0 55)*
(0.54)*
(0.43)*
(0.5)*
(0.40)
(0 37)
(0.50)*
(0.33)*
Day 44
4.91
4.14
2.31
2.08
1.91
(0.21)*
(0.28)
(0.15)*
(0.12)*
(0.13)*
17.6
7.75" (0.93)
5 48 (0 61)
1.95 (0.19)*
42.8
5 32 (0.71)*
5.59 (0.45)
1.71 (0.14)*
127
127
3 22 (0.27)*
5 09* (0 57)*
4.06 (0.36)
3.93 (0.57)*
1.66C (0.17)*
127
3 67* (0 32)*
3.77 (0.25)*
17 6
5.39 (0.46)*
4.77 (0.39)
42 8
8.89 (0.54)
5.53 (0.52)
6.75* (0 68)*
6.01 (0.56)
2.44 (0.13)*
2.19
(0.22)*
3.22 (0.16)*
2.65 (0.18)*
127
HI vs LO (LA + CS)
HI (LA) vs LO (LA + CS)
ME vs LO (LCS + CS)
HI (LCS) vs HI (LS + CS)
HI (LCS) vs HI (LA)
HI (LCS) vs HI (LA + CS)
HI
HI
HI
HI
HI
HI
(LA) vs LO (LA + CS)
(LA) vs ME (LA + CS)
(LCS) vs HI (LS)
(LCS) vs HI (LS + CS)
(LCS) vs HI (LA)
(LCS) vs HI (LA + CS)
2.63 (0.15)*
HI (LCS) vs HI (LA + CS)
° Values are mean activity in U/liter of n = 10 per group except where indicated. Standard errors (SE) are in parentheses. All groups for a specific
day are significantly different from the control (blank feed) for that same day.
* Groups for which n = 9.
c
Groups for which n = 8.
d
LA, lead acetate; CS, control soil; LS, lead sulfide; LCS, lead-contaminated soil.
* Significantly different from control soil (1.5%) group at the 0.05 level (Dunnett's test).
study. In contrast, lead concentrations in bone from animals
receiving control soil were approximately two to five times
higher and increased at the later time points. However, significant differences between both control groups, control
feed and feed plus soil, and treated groups were evident for
all groups at all time points.
As anticipated, the highest concentrations of lead in bone
were observed in animals receiving lead acetate. In these animals, lead concentrations in bone increased in both a doseand time-dependent manner for all groups. At 15 and 44 days,
the addition of control soil with lead acetate resulted in lower
lead levels in bone. Lead concentrations in bone were still
dose-dependent, but contrary to the observations with lead
acetate alone, there were no significant increases in lead in
bone with time when control soil was included in the diet.
Administration of lead sulfide in the diet resulted in only
20 to 30% of the bone lead concentrations achieved following
administration of similar dose of lead acetate but, as observed
with lead acetate, lead levels increased with the time of exposure. The addition of control soil to the diet with lead sulfide
resulted in significantly increased bone lead levels at 7 days,
but had little or no significant effect at the later time points.
Administration of lead-contaminated soil resulted in significantly increased bone lead, but the increases were less than
observed with the lead salts. Bone lead concentrations and
increases in concentrations with time were significantly less
when lead was administered in contaminated soil. Concentrations above background levels tended to increase with time,
but the increases were frequently not significant. The most
significant increase between the low and the two higher doses
was observed at 44 days.
Liver and Kidney
In addition to the determination of lead in blood and bone,
this study also determined the accumulation of lead in liver
114
FREEMAN ET AL.
TABLE 4
Group Mean Blood Lead Concentrations
Mean (SEf
Dose group
Control (blank feed)
Control soil (1.5%)
Lead acetate (LO)
Lead acetate (ME)
Lead acetate (HI)
Lead acetate + control soil
(1.5%) (LO)
Lead acetate + control soil
(1.5%) (ME)
Lead acetate + control soil
(1.5%) (HI)
Lead sulfide (HI)
Lead sulfide + control soil
(1.5%) (HI)
Lead-contaminated soil (0.2%)
+ control soil (1.3%) (LO)
Lead-contaminated soil (0.5%)
+ control soil (1.0%) (ME)
Lead-contaminated soil (1.5%)
(HI)
Pairs of noncontrol dose groups
significantly different at 0.05
level (Tukey's test on log
concentrations)''
Day 7
ppm
<l
17.6
42.8
127
0 050
0.113
0.320
0 376
0 771
Day 44
Day 15
0.027 (0.003)*
0.102* (0.009)
0.205 (0.016)*
0.424 (0.084)*
0.676 (0 081)*
(0.009)*
(0.018)
(0.069)*
(0.054)*
(0.112)*
0.012
0.033
0.249
0.405
(0.001)*
(0.004)
(0.019)*
(0.028)*
0.706 (0.034)*
176
0.254 (0.052)*
0 143 (0014)
42.8
0.384 (0.060)*
0.143 (0.010)
127
127
2.08 (0.29)*
0.241 (0015)*
0.380 (0 049)*
0.245 (0 028)*
0.442c (0.042)*
0.264 (0.024)*
127
0.501 (0.111 )*
0.230 (0.026)*
0 296 (0.023)*
17.6
0.206 (0.016)*
0.157 (0016)
0.122 (0.007)*
42.8
0.152 (0.017)
0.166 (0.024)
0.169 (0.020)*
0.190 (0.020)*
0.137 (0.030)
0.166 (0.013)*
HI
HI
HI
HI
HI
HI
HI
HI
HI
HI
HI
HI
HI
HI
HI
HI
HI vs LO (LA)
HI vs ME (LA)
ME vs LO (LA)
HI vs LO (LA + CS)
HI vs ME (LA + CS)
HI (LA) vs HI (LA + CS)
HI (LA) vs ME (LA + CS)
HI (LA) vs LO (LA + CS)
ME (LA) vs ME (LA + CS)
ME (LA) vs LO (LA + CS)
LO (LA) vs HI (LA + CS)
HI (LA + CS) vs HI (LCS)
HI (LA) vs HI (LS)
HI (LA) vs HI (LS + CS)
HI (LA) vs HI (LCS)
HI (LS) vs HI (LCS)
HI (LS + CS) vs HI (LCS)
HI vs LO (LA)
HI vs ME (LA)
ME vs LO (LA)
HI vs LO (LA + CS)
HI vs ME (LA + CS)
HI (LA) vs HI (LA + CS)
HI (LA) vs ME (LA + CS)
HI (LA) vs LO (LA + CS)
ME (LA) vs LO (LA + CS)
ME (LA) vs ME (LA + CS)
LO (LA) vs HI (LA + CS)
HI (LA + CS) vs HI (LS)
HI (LA + CS) vs HI (LCS)
HI (LA) vs HI (LS)
HI (LA) vs HI (LS + CS)
HI (LA) vs HI (LCS)
HI (LS + CS) vs HI (LCS)
0.164 (0.013)*
0 193 (0.017)*
127
vs LO (LA)
vs ME (LA)
vs LO (LA + CS)
vs ME (LA + CS)
(LA + CS) vs LO (LA)
(LA + CS) vs ME (LA)
(LA + CS) vs HI (LA)
(LA) vs ME (LA + CS)
(LA) vs LO (LA + CS)
(LA + CS) vs HI (LS + CS)
(LA + CS) vs HI (LS)
(LA + CS) vs HI (LCS)
(LA) vs HI (LS)
(LA) vs HI (LCS)
(LS + CS) vs HI (LS)
(LS + CS) vs HI (LCS)
° Values are mean concentrations in fjg Pb/g of n = 10 per group except where indicated Standard errors (SE) are in parentheses. All groups for a
specific day are significantly different from the control (blank feed) for that same day.
* Groups for which n = 9.
' Groups for which n = 8.
''LA. lead acetate; CS. control soil: LS. lead sulfide: LCS, lead-contaminated soil.
* Signficantly different from control soil (1.5%) group at the 0.05 level (Dunnett's test on log concentrations).
and kidney of animals from each of the treated groups. For
the sake of brevity, these data are not presented, but are
briefly described below.
Concentrations of lead seen in liver were similar to those
in blood. Background levels in animals receiving blank feed
ranged from 0.232 to 0.009 ng Pb/g and, as with blood, were
lowest at the later time points. Administration of control soil
resulted in zero to six times higher lead levels than seen in
the control feed animals. Liver lead levels tended to be lower
at the later time points, but the trend was not significant.
Liver levels achieved with lead acetate were dose-dependent,
and the highest levels were seen at Day 7 with no significant
change thereafter. Coadministration of control soil with lead
acetate generally resulted in lower liver levels, but the effect
was frequently not significant. Lead sulfide administration
resulted in liver lead levels that were significantly above
115
EVALUATION OF LEAD-CONTAMINATED SOILS
TABLE 5
Group Mean Bone Lead Concentrations
Mean (SE)°
Dose group
Control (blank feed)
Control soil (1.5%)
Lead acetate (LO)
Lead acetate (ME)
Lead acetate (HI)
Lead acetate + control soil
(1.5%) (LO)
Lead acetate + control soil
(1.5%) (ME)
Lead acetate + control soil
(1.5%) (HI)
Lead sulfide (HI)
Lead sulfide + control soil
(1 5%) (HI)
Lead-contaminated soil (0.2%)
+ control soil (1.3%) (LO)
Lead-contaminated soil (0.5%)
+ control soil (1 0%) (ME)
Lead-contaminated soil (1.5%)
(HI)
17.6
42.8
127
3.69
6.43
36 0
45.8
105
(0.96)
(1.57)
(9.6)*
(6 8)*
(14)*
3.38
12.9
42.3
110
167
17.6
25.4 (6.6)*
42 8
50.6 (9.9)*
127
127
Day 15
Day 7
ppm
252 (27)*
20.4 (1.9)*
(0.50)*
(1.6)
(5.3)*
(27)*
(22)*
Day 44
2.23 (0.24)*
9.99(1.70)
73.9 (7.6)*
174
(18)*
374
(29)*
29.6 (4.7)*
25.7 (1.9)*
33 2 (2 7)*
45.0 (3.8)*
120
(16)*
(27)*
74.8 (16.9)*
46.7
17.6
16.7 (2.0)*
54.7 (9 3)*
89.8* (10.8)*
42.8
13.8 (1.8)*
24.9 (3.9)*
27.3 (1.8)*
20.0 (3.4)*
32 9 (8.4)*
51.5 (8 3)*
127
127
Pairs of noncontrol dose groups
significantly different at 0.05
level (Tukey's test on Log
concentrations)''
(6.2)*
189
67.7 (8.8)*
22.5 (4.6)
50.8 (8.6)*
HI vs LO (LA)
HI vs ME (LA)
ME vs LO (LA)
HI vs LO (LA)
HI vs LO (LA)
HI vs LO (LA + CS)
HI vs ME (LA)
ME vs LO (LA)
HI vs ME (LA + CS)
HI vs LO (LA + CS)
HI vs LO (LA + CS)
ME vs LO (LA + CS)
HI vs ME (LA + CS)
HI vs ME (LA + CS)
ME vs LO (LCS + CS)
ME vs LO (LA + CS)
HI (LA) vs LO (LA + CS)
LO (LA) vs LO (LA + CS)
HI (LA) vs LO (LA + CS)
HI (LA) vs ME (LA + CS)
LO (LA) vs HI (LA + CS)
ME (LA) vs ME (LA + CS)
HI (LA) vs ME (LA + CS)
ME (LA) vs LO (LA + CS)
HI (LA) vs HI (LA + CS)
ME (LA) vs LO (LA + CS)
ME (LA) vs ME (LA + CS)
HI (LA + CS) vs LO (LA)
HI (LA + CS) vs LO (LA)
HI (LA) vs LO (LA + CS)
HI (LA + CS) vs ME (LA)
HI (LA) vs HI (LS)
HI (LA) vs ME (LA + CS)
HI (LS + CS) vs HI (LS)
HI (LA) vs HI (LS + CS)
HI (LA) vs HI (LA + CS)
HI (LA) vs HI (LS)
HI (LA) vs HI (LCS)
HI (LS + CS) vs HI (LCS)
HI (LA) vs HI (LCS)
HI (LA + CS) vs HI (LS)
HI (LA) vs HI (LS)
HI (LA + CS) vs HI (LS)
HI (LA + CS) vs HI (LS + CS)
HI (LA) vs HI (LS + CS)
HI (LA + CS) vs HI (LS + CS)
HI (LA + CS) vs HI (LCS)
HI (LA) vs HI (LCS)
HI (LA + CS) vs HI (LCS)
HI (LS) vs HI (LCS)
HI (LA + CS) vs HI (LS)
HI (LS + CS) vs HI (LCS)
HI (LS + CS) vs HI (LCS)
HI (LA + CS) vs HI (LS + CS)
HI (LA + CS) vs HI (LCS)
" Values are mean concentrations in nf> Pb/g of n = 10 per group. Standard errors (SE) are in parentheses. All groups for a specific day are significantly
different from the control (blank feed) for that same day except for the control soil (1.5%) group for Day 7.
* LA, lead acetate; CS, control soil; LS, lead sulfide; LCS, lead-contaminated soil
* Significantly different from control soil (1.5%) group at the 0.05 level (Dunnett's test on log concentrations).
background, but also only about 25 to 35% of those seen
with similar doses of lead acetate. As with blood, coadministration of control soil had little effect on the absorption of
lead sulfide. Among animals receiving lead, the lowest concentrations of lead in liver were seen following administration of lead-contaminated soil, and these animals tended to
have the highest concentrations in liver at Day 7.
Lead accumulation in the kidney is second only to bone
and with high doses of lead results in unique morphological
changes (Fowler et al, 1980; Vyskocil et ai, 1989). The
present study was no exception in that lead levels in kidneys
of blank feed animals ranged from 0.551 to 0.124 pig Pb/g
with the highest concentrations seen at Day 7 and lower
levels seen at the later time points. Addition of control soil
116
FREEMAN ET AL.
TABLE 6
Relative Bioavailability Values of Lead in Soil versus
Soluble and Relatively Insoluble Lead Salts
Ratio of (lead in body)/
(lead in diet)
Treatment group
Day 7
Day 15
Day 44
Lead acetate (LO)
Lead acetate (ME)
Lead acetate (HI)
Lead acetate + control soil (LO)
Lead acetate + control soil (ME)
Lead acetate + control soil (HI)
Lead sulfide (HI)
Lead sulfide + control soil (HI)
Lead-contaminated soil + control
soil (LO)
Lead-contaminated soil + control
soil (ME)
Lead-contaminated soil (HI)
0.251
0.151
0.156
0.128
0.117
0.169
0.014
0.050
0.165
0.182
0.139
0 086
0.046
0.046
0017
0.020
0.134
0.143
0.139
0 037
0.028
0.034
0.012
0.016
0.087
0.071
0.037
0.028
0.014
0 036
0.008
0.027
0.0O9
to the diet resulted in 2.5- to 5-fold higher lead concentrations in kidney than seen in animals receiving blank feed.
Lead in kidneys of most animals receiving control soil
trended lower at the later time points. Of the lead sources
administered, lead acetate resulted in the highest kidney concentrations. Lead in kidney generally increased in a doseand time-dependent manner. The addition of control soil in
the diet with lead acetate significantly attenuated the accumulation of lead in kidney and abolished the time-dependent
effect so that the highest concentrations were seen at Day
7. Dose-dependent accumulation of lead in kidney following
administration of lead acetate with control soil was still significant. Lead sulfide or lead sulfide plus control soil resulted
in approximately 30 to 35% of the lead concentrations
achieved with lead acetate and the maximum concentrations
were seen by Day 7 with little to no significant changes
thereafter. The presence of control soil had little or no effect
on the accumulation of lead in kidney following administration of lead sulfide. Animals receiving lead-contaminated
soil tended to have higher concentrations of lead in kidney
than animals receiving control soil, but the differences were
not significant.
Bioavailability
Bioavailability of each of these forms of lead was calculated by using the formula described by O'Flaherty (1991,
1995). These calculations were based on the assumption that
the amount of lead in the tissues for which lead was not
determined in this study was 0.04 times the total actually
determined in blood, bone, liver, and kidney. Results of these
calculations are presented in Table 6. As anticipated, the
data indicate that the more soluble lead salt, lead acetate,
was much more readily absorbed and retained than either
lead sulfide or lead from contaminated soil. With exception
of the low dose at Day 7 which appears somewhat out of
the expected range, approximately 14 to 17% of the lead
acetate in the diet was absorbed and retained. The data also
indicate that these numbers were relatively constant across
the dose range and at all times assayed. The addition of
control soil to the diet with lead acetate resulted in a significant decrease in lead bioavailability. Further, the availability
of lead from lead acetate plus control soil appeared to decrease as the dose increased and as the time of feeding
increased.
Lead sulfide was only approximately 10% as available as
lead acetate, but, as observed with lead acetate, the bioavailability of lead sulfide appeared relatively constant with time.
With exception of the possibly spurious data at Day 7 that
indicate the addition of control soil to the diet with lead
sulfide increased bioavailability, control soil had little effect
on the bioavailability of lead from lead sulfide. The bioavailability of lead from contaminated soil varied considerably
with dose and time. In general, bioavailability appeared to
be the inverse of the dose administered and decreased with
the time of exposure.
DISCUSSION
Oral consumption of lead carried on dust and dirt originating from contaminated soil encountered in the environment
is the major source of lead exposure for many children.
However, it appears that not all exposures to lead pose the
same risk. That is, lead bioavailability can vary greatly with
the form and matrix in which lead is received. This fact is
evidenced in the human population by the fact that lead
levels detected in the blood of exposed children are frequently not proportional to the total lead in their environment
or their estimated level of ingestion. Given this fact, it is
unlikely that a single default assumption of lead bioavailability would be appropriate to assess risks associated with all
environmental sources of lead exposure. It would seem more
reasonable to develop an estimate of lead bioavailability
from the source of contamination in question. This is particularly the case when one considers that assessments of risk
associated with lead exposure may have profound socioeconomic ramifications including removal of large amounts of
residential soil and disruption of the lives of the people
involved. Therefore, the present study was designed to address three objectives: (1) to determine lead bioavailability
from actual contaminated soil versus lead salts; (2) to determine if the bioavailability of lead salts is altered by the
presence of soil in the diet; and (3) to evaluate the weanling
rat as an animal model for use in relatively rapid and economical determinations of lead bioavailability from specific
sites.
EVALUATION OF LEAD-CONTAMINATED SOILS
Results of these studies indicate that control soil alone
can have an effect on A-ALAD activity in very young rats.
The source of this effect is not known. Metals which reverse
the A-ALAD inhibiting effects of lead include zinc (Johnson
and Tenuta, 1979; Markowitz and Rosen, 1981), cadmium
(Davis and Avran, 1978), copper (Klauder and Petering,
1975; Underwood, 1977), and iron (Bota ei al., 1982). Lead
inhibition of A-ALAD activity can be enhanced by restrictions in dietary calcium, iron, phosphorous, vitamins A and
D, and protein (ATSDR, 1991). However, the similar body
weight gain between the control blank feed and control soil
groups argues against any impaired absorption of nutrients
that would lead to enhanced absorption of lead present in
the soil. Therefore, we are unaware of anything in the control
soil other than the traces of lead that could have inhibited
A-ALAD.
As anticipated, administration of lead acetate resulted in
the greatest inhibition of A-ALAD. A-ALAD inhibition by
lead acetate was attenuated by the addition of control soil
to the diet. This effect is speculated to be due to the calcium
and/or other minerals in the soil, but a similar effect was
not observed with lead sulfide. Lead acetate with or without
soil resulted in greater decreases in A-ALAD than lead sulfide and A-ALAD was inhibited least by lead-contaminated
soil. The relationship of increased inhibition with increasing
dose that was evident for the lead acetate or lead acetate/
control soil groups did not hold for lead-contaminated soil.
The control blank feed group, theoretically free from dietary and drinking water lead, exhibited steadily decreasing
blood and bone lead concentrations from Days 7 to 44. Plausible explanations for this observation are a change in the
dietary lead exposure when the rats were put on study and
their rapid growth while on study. Also, prior to arrival at
the laboratory, the rats (3 weeks of age upon arrival) were
nourished by maternal milk and/or in-house feed which most
likely exceeded the concentration of lead in the AIN-76A
feed given to the animals after they arrived. Concentrations
of lead in blood and bone at any given time period were,
in most instances, significantly greater for the control soil
treatment compared to the control blank feed group. These
results indicate that, in this model, lead in the soil at concentrations as low as 0.4 ppm was absorbed sufficient to increase
blood and bone lead to levels that could be measured and
distinguished from background concentrations.
Morgan etal. (1977) reported the half-life of lead in blood
following intravenous administration was 280 hr. Since
steady state is reached in approximately four half-lives, 46
days, it is assumed that lead concentrations observed in tissues, other than bone, in this study were at approximate
steady-state levels. Bone lead concentrations did not indicate
a biphasic change in lead concentration over time but, for
most treatment and dose groups, lead concentrations in bone
continued to increase up through Day 44. This finding is
117
consistent with the well-established incorporation of lead
into bone and, thus, represents lead that is not freely exchanged. The half-life of this very-slow phase of distribution,
where lead is distributed between the bone and blood, has
been estimated to be as long as a decade in some species
(Lloyd et al, 1975).
Results presented here demonstrate that the soil matrix
used attenuates the bioavailability of one lead salt, lead acetate, while having minimal effect on the bioavailability of
another, lead sulfide. The source of this variability is not
known, but it is known that the most critical factor governing
absorption of lead from the gastrointestinal tract is the rate
of dissolution. The capacity of control soil to impair absorption of lead acetate might be explained by its effect on the
solubilization process. The presence of 1.5% soil in the diet
is thought to have minimal effect on gastric/intestinal pH or
gastric emptying, but other minerals in soil could possibly
interact with lead to slow dissolution. The lack of an effect
on the bioavailability of lead sulfide indicates the effect of
control soil on lead acetate is not due to an effect on gastric
emptying and may indicate that chemical interactions with
the salt or modulation of gastric pH are not involved. Most
importantly, results presented here indicate that lead consumed with soil is unlikely to be more bioavailable than the
original lead salt contained in soil and may be significantly
less available than the forms of lead on which current default
assumptions are based. The low bioavailability of lead from
contaminated soil may be due to the particle size used, other
minerals in soil and/or the "armoring" process typical of
soil lead. Both particle size and armoring have been reported
to greatly inhibit lead dissolution (Freeman et al., 1992,
1994).
In conclusion, results of this study demonstrate that the
immature rat can be used as a very sensitive indicator of
lead bioavailability from a variety of sources including contaminated soil. Quantifiable effects seen with the very low
levels of lead in control soil indicate the model has potential
to detect the effect of lead at levels significantly below the
17 ppm level used in this study. The rapid response of blood
lead levels and A-ALAD indicates that the assay might be
used to determine lead bioavailability from contaminated
soil in a relatively short period of time. These results also
indicate that the presence of one or more components of soil
used in this study may attenuate the bioavailability of one
form of lead, while having minimal effect on others. Taken
together these results indicate that some assay of lead bioavailability from individual contaminated sites, particularly
soil from residential areas, should be used as part of any
assessment of associated human health risks.
ACKNOWLEDGMENTS
The authors express their appreciation to Ms. Diana Reichelderfer, Ms.
Cynthia Rivera-Snyder, and Mr A. Dale Marcy for their excellent technical
118
FREEMAN ET AL.
assistance. This work was supported by the National Toxicology Program.
The work of Dr. Parham was supported by the Agency for Toxic Substances
and Disease Registry.
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